Inhibition of let7i as a means to enhance the protective effect of progesterone against stroke

ABSTRACT

The subject invention provides methods of treating neurological disease or disorder, such as brain injuries, such as stroke, traumatic brain injury (TBI), or other ischemic events that cause brain injury by inhibiting or down-regulating Let-7i activity or function. The disclosed methods may have the potential to extend the “window of opportunity” for treatment of such injuries and enhance the effectiveness of existing therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/639,139, filed Feb. 14, 2020, now U.S. Pat. No. 11,230,711, which isthe U.S. National Stage Application of International Patent ApplicationNo. PCT/US2018/046456, filed on Aug. 13, 2018, which claims the benefitof U.S. Provisional Application Ser. No. 62/544,994, filed Aug. 14,2017, the disclosure disclosures of which is are hereby incorporated byreference in its their entirety, including all figures, tables and aminoacid or nucleic acid sequences.

GOVERNMENT SUPPORT

This invention was made with Government support under AG027956 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Aug. 13, 2018 and is 1 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Stroke is the fourth leading cause of death and a major cause ofdisability in the US [14], costing approximately $34 Billion annually(according to the Center for Disease Control). The risk of ischemicstroke dramatically increases with age. Of note, the incidence ofischemic stroke is relatively rare among pre-menopausal women [1].Although middle aged women have a lower risk of stroke than men, strokebecomes more prevalent in post-menopausal women compared to men of thesame age [1]. With increasing age, circulating gonadal hormone levelsdecline in both males and females, however, such age-associateddecreases are much more dramatic in women, and is a function of themenopause. While much attention has been placed on the loss of estrogenfollowing the menopause, it is worth noting that the levels of P4 alsodecline precipitously. As such, the increased risk for stroke inpostmenopausal women may be due to a decline in not just estrogenlevels, but that of P4 as well. In fact, growing literature hassuggested that P4 is protective, and is (neuro)protective in a varietyof experimental models of stroke [2-4]. However, the underlyingmechanisms for P4's protective effects remain unclear. It is thisincomplete information that limits our understanding of why P4'sbeneficial effects were equivocal in the latest Phase III clinical trialof P4 efficacy in treating traumatic brain injury, despite numerousother studies (both preclinical and clinical) that demonstrate itspositive efficacy. We suggest that a better understanding of the factorsthat influence the expression of key mediators (e.g., receptors) of P4is critical to advancing the development of effective P4-basedneuroprotectants.

It is also worth pointing out that the literature associated with P4'sprotective effects has focused on a direct effect of P4 on neurons. Thenotion that glia may be an equally important target underlying P4'sprotective effects on the brain has only been studied minimally. Indeed,astrocytes have been considered as an important component in thepost-ischemic recovery, as these cells are critical for regeneration andremodeling of neural circuits following stroke [9].

A known mediator of P4's neuroprotective action is brain-derivedneurotrophic factor (BDNF) [15]. A deficit in BDNF has been linked tostroke pathophysiology [16, 17]. In the central nervous system (CNS),BDNF also has an established role in promoting neuronal differentiation,survival, synaptic plasticity [6-8] and synaptogenesis [18-20].Synaptogenesis occurring in the penumbra is known to strongly contributeto enhanced functional recovery from stroke [21-24]. Based on theseobservations, it is plausible that the P4/BDNF signaling-mediatedenhanced synaptogenesis and neuroprotection may contribute to P4'sprotective effects during post stroke brain repair. We recently reportedthat P4 elicits the release of BDNF from primary astrocytes via aputative membrane progesterone receptor consisting ofprogesterone-receptor-membrane-component-1 (Pgrmc1) [10]. Our resultssuggest that conditioned medium derived from P4-treated astrocytes, whenapplied to primary cortical neurons, increases the expression ofsynaptic markers in these neural cells and enhances their survivalagainst oxidative stress. Our data support the model whereby P4 elicitsits (neuro)protective effects through a mechanism that involvesPgrmc1-dependent BDNF release from glia.

Currently, knowledge regarding the regulation of Pgrmc1 in brain and theconsequence of such regulation is limited. Studies from our labdemonstrate that the miRNA, let-7i, negatively regulates expression ofboth Pgrmc1 and BDNF in glia, leading to suppression of P4-induced BDNFrelease from glia and attenuation of P4's beneficial effects onneuroprotection and synaptogenesis in the ischemic brain. Furthermore,the increased expression of let-7i with stroke may explain why poststroke therapy may not be so effective. As there remains a significantneed for treatments of brain injuries, such as stroke and traumaticbrain injury (TBI), down-regulation of let-7i may have the potential toextend the “window of opportunity” for treatment of such injuries.

BRIEF SUMMARY OF THE INVENTION

The neuroprotective effects of P4 have been reported since 1996 [25],however, knowledge of what governs the protective effects of P4 is stilllargely lacking. Further, a heavy emphasis has been placed on P4's“genomic” mechanism(s) of action, elicited via the classicalprogesterone receptor (PR), and that too, focused on neurons. However,evidence from emerging literature as well as from our own recent studieshave highlighted the critical role of glia, both as a site of local P4synthesis and as a mediator of P4's pro-survival functions in CNS[26-28]. The subject invention provides methods of treating neurologicaldisease or disorder, such as brain injuries, such as stroke, traumaticbrain injury (TBI), or other ischemic events that cause brain injury byinhibiting or down-regulating Let-7i activity or function. The disclosedmethods may have the potential to extend the “window of opportunity” fortreatment of such injuries and enhance the efficacy of existingtreatments for such injuries.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1B: Conditioned medium derived from P4 (10 nM)-treatedastrocytes (P4-ACM, 18 hrs) and BDNF (50 ng/ml, 18 hrs) increasedexpression of Synaptophysin and Gap-43 in primary cortical neurons. FIG.1A represents immune-staining of Gap43 (green) and Synaptophysin (red).FIG. 1B represents qRT-PCR analysis of Gap43 (left) and Synaptophysin(right) mRNA levels. Data are presented as a percentage of control(non-treated group) (*:p≤0.05, **:p≤0.01).

FIGS. 2A-2B: Over-Expression of Let-7i down-regulated Pgrmc1 and BDNFmRNA in primary astrocytes. Cells were transfected with let-7i or let-7fmimic or a negative control 48 hrs prior to RNA isolation for qRT-PCR.Quantification of Pgrmc1 and BDNF mRNA levels was normalized to GAPDH(FIG. 2A). Quantification of let-7 levels was normalized to U6 snRNA(FIG. 2B). Data are presented as a percentage of control (***:p≤0.001,n.s: non-significant).

FIG. 3: Let-7i attenuates P4-induced BDNF release from primary corticalastrocytes, measured by in-situ BDNF ELISA (***:p≤0.001, n.s:non-significant).

FIG. 4: Age-related decrease of Pgrmc1 expression correlated withdecrease of BDNF in mouse hippocampus. mRNA levels were measured byqRT-PCR (young: 6-mo old; middle-aged: 12-mo old; old: 24-mo old. Datapresented as a percentage of young group (*:p≤0.05, **:p<0.01, n.s:non-significant).

FIGS. 5A-5B: A decrease of Pgrmc1 expression correlates with an increaseof Let-7i in cortex at day 7 post-stroke. Total RNA was measured byqRT-PCR. Pgrmc1 mRNA was normalized to GAPDH (FIG. 5A). Let-7iexpression was normalized to U6 snRNA (FIG. 5B) (*:p≤0.05).

FIG. 6: Ischemic injury is greatly reduced in animals receiving P4 andthe Let-7i inhibitor.

FIG. 7: Functional recovery (grip strength) is greatly enhanced inanimals receiving ICV injections of the Let-7i inhibitor.

FIGS. 8A-8B: Oxygen-Glucose Deprivation (OGD) results in an increase inlet-7i expression and suppresses progesterone (P4)-induced BDNF releasefrom primary cortical astrocytes. Primary cortical astrocytes wereexposed to one-hour of OGD. Immediately after re-instatement of normaloxygen and glucose concentrations, these cells were either mocktransfected (control) or transfected with the let-7i antagomir. 12 hourslater, expression of let-7i was evaluated (FIG. 8A) (n=4). n.s: notsignificant, ****P<0.0001 compared to mock transfected control (mock).(FIG. 8B) Quantitation of BDNF release measured by BDNF in situ ELISA(n=4). n.s: not significant compared to DMSO group. Data are presentedas mean±SEM.

FIG. 9: let-7i prevents progesterone (P4)-induced neuroprotectionagainst oxygen-glucose-deprivation (OGD). Conditioned-media derived fromhormone or control-treated astrocytes were applied to primary corticalneurons (DIV 14) after one-hour exposure to OGD. BDNF (50 ng/ml) wasdirectly added to neurons after OGD to serve as positive control.Neuronal viability was measured by CellTiter-Glo viability assay (n=5).n.s: not significant, ***P<0.001 and **P<0.01 compared to normoxia. Dataare presented as the mean±SEM.

FIGS. 10A-10C. let-7i inhibits progesterone (P4) induces synaptophysin(SYP) expression in primary cortical neurons. (FIG. 10A) Representativeconfocal images of primary cortical neurons (DIV 14) immunostained withsynaptophysin (SYP, green) and DAPI (blue). (60x, Scale bars=30μm).(FIG. 10B) Quantification of average number of SYP puncta per neuron(n=3). n.s: not significant, ***P<0.001 compared to mock transfected+DMSO group. (FIG. 10C) Representative immunoblots probed for SYP andquantification graph of relative SYP protein ratio to Gapdh (n=4). n.s:not significant, ****P<0.0001, ***P<0.001 compared to mocktransfected+DMSO group. Data are presented as the mean±SEM.

FIGS. 11A-11E: Combined treatment with progesterone (P4) and the let-7iinhibitor reversed ischemia-induced suppression of Pgrmc1 and BDNFexpressions in the penumbra. (FIG. 11A) Representative immunoblotsprobed for Pgrmc1, pro- and mature-BDNF. (FIG. 11B) Quantitation graphof relative Pgrmc1 protein ratio to Gapdh (n=4-5 per group). (FIG. 11C)Quantitation graph of relative pro-BDNF protein ratio to Gapdh (n=4-5per group). (FIG. 11D) Quantitation graph of relative mature BDNFprotein ratio to Gapdh (n=4-5 per group). (FIG. 11E) Quantitation graphof relative let-7i expression in ischemic brain (n=4-5 per group). n.s:not significant, ** P<0.01 and *P<0.05 compared to sham, and #P<0.05compared to P4+ scrambled. Data are presented as the mean±SEM.

FIGS. 12A-12B: Co-administration of let-7i antagomir (anti-let-7i) andprogesterone (P4) reduces ischemic injury. (FIG. 12A) Representativeimages of serial coronal brain sections stained withtriphenyltetrazolium chloride (TTC). (FIG. 12B) Quantification ofinfarct sizes of TTC-stained images (n=4 per group). n.s: notsignificant, ***P<0.001 and **P<0.01 compared to cholesterol+scrambledgroup. Data are presented as the mean ±SEM.

FIG. 13: Co-administration of let-7i antagomir (anti-let-7i) andprogesterone (P4) enhances recovery of motor function/grip strengthfollowing stroke. Results of wire suspension test at day 3, 7 and 14post stroke (n=15-20 per group). n.s: not significant, ***P<0.001 and **P<0.01 compared to sham, ###P<0.001, ##P<0.01 compared to P4+ scrambled,and $$P<0.01 compared to cholesterol+scrambled. Data are presented asthe mean ±SEM.

FIGS. 14A-14D: Inhibition of let-7i enhances progesterone (P4)'s effecton the expression of synaptophysin in the penumbra. (FIG. 14A)Representative confocal images of penumbra region staining forsynaptophysin (SYP, red) and DAPI (blue). (60×, Scale bars=30 μm). (FIG.14B) Quantification of average relative SYP puncta presents in eachfield (n=3 per group). n.s: not significant and ***P<0.001 compared tosham, ###P<0.001 and ##P<0.01 compared to P4+ scrambled. (FIG. 14C)Representative immunoblots probed for SYP protein. (FIG. 14D)Quantification graph of Syp signal, expressed as a ratio to Gapdh (n=4-5per group). n.s: not significant, ***P<0.001 and **P<0.01 compared tosham, ##P<0.01 and #P<0.05 compared to P4+ scrambled.

DETAILED DISCLOSURE OF THE INVENTION

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value.

As used herein, the “Let-7i” sequence comprises:

mmu-let-7i-5p MIMAT00001225′ UGAGGUAGUAGUUUGUGCUGUU 3′ (SEQ ID NO: 1). The let-7i-5p sequence isidentical for both the human (MIMAT0000415) and murine (MIMAT0000122)miRNA. The full length human and murine let-7i sequences, including thestem loop can be obtained at the miRBase database (mirbase.org) asaccession numbers MI0000434 (human, SEQ ID NO: 2) and MI0000138 (murine,SEQ ID NO: 3).

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA. An antisense oligonucleotide can upregulate or downregulateexpression and/or function of a particular polynucleotide. Thedefinition is meant to include any foreign RNA or DNA molecule which isuseful from a therapeutic, diagnostic, or other viewpoint. Suchmolecules include, for example, antisense RNA or DNA molecules,interference RNA (RNAi), short hairpin RNA (shRNA), and silencing RNA(siRNA). Inhibitory oligonucleotides and vectors for deliveringinhibitory oligonucleotides for Let-7i are commercially available fromvendors such as Vigene Biosciences, Inc. (Rockville, Md. 20850 USA),OriGene Technologies, Inc. (Rockville, Md. 20850 USA), and Santa CruzBiotechnology, Inc. (Dallas, Tex. 75220 USA).

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucleosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, Hoogsteen orreverse Hoogsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide inthe case of an oligonucleotides compound. These oligonucleotidestypically comprise at least one region wherein the oligonucleotide ismodified in order to exhibit one or more desired properties. The desiredproperties of the oligonucleotide include, but are not limited, forexample, to increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. Different regions of the oligonucleotide may thereforehave different properties. The chimeric oligonucleotides of the presentinvention can be formed as mixed structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide analogs as described above.

The oligonucleotide can be composed of regions that can be linked in“register”, that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in preferredcases a length not exceeding about 100 carbon atoms. The spacers maycarry different functionalities, for example, having positive ornegative charge, carry special nucleic acid binding properties(intercalators, groove binders, toxins, fluorophores, etc.), beinglipophilic, inducing special secondary structures like, for example,alanine containing peptides that induce alpha-helices.

As used herein “BDNF” and “Brain derived neurotrophic factor” areinclusive of all family members, mutants, alleles, fragments, species,coding and noncoding sequences, sense and antisense polynucleotidestrands, etc. As used herein, the terms “Brain derived neurotrophicfactor”, “Brain-derived neurotrophic factor” and BDNF, are consideredthe same in the literature and are used interchangeably in the presentapplication.

“Progesterone” includes all natural forms of progesterone as well aschemically synthesized analogs of progesterone.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene (in this case let-7i), or (ii) capable of forming a stableduplex with a portion of a mRNA transcript of the targeted gene.Stability of the complexes and duplexes can be determined by theoreticalcalculations and/or in vitro assays. Exemplary assays for determiningstability of hybridization complexes and duplexes are described in theExamples below.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences (in this case let-7i). In certain embodiments of thepresent invention, the mediators are 5-25 nucleotide “small interfering”RNA duplexes (siRNAs). The siRNAs are derived from the processing ofdsRNA by an RNase enzyme known as Dicer. siRNA duplex products arerecruited into a multi-protein siRNA complex termed RISC (RNA InducedSilencing Complex). Without wishing to be bound by any particulartheory, a RISC is then believed to be guided to a target nucleic acid(suitably mRNA), where the siRNA duplex interacts in a sequence-specificway to mediate cleavage in a catalytic fashion. Small interfering RNAsthat can be used in accordance with the present invention can besynthesized and used according to procedures that are well known in theart and that will be familiar to the ordinarily skilled artisan. Smallinterfering RNAs for use in the methods of the present inventionsuitably comprise between about 1 to about 50 nucleotides (nt). Inexamples of non-limiting embodiments, siRNAs can comprise about 5 toabout 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15to about 25 nt, or about 20-25 nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

The term “nucleotide” covers naturally occurring nucleotides as well asnon-naturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof. Illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in Benner et al., U.S. Pat. No. 5,432,272. The term“nucleotide” is intended to cover every and all of these examples aswell as analogues and tautomers thereof. Especially interestingnucleotides are those containing adenine, guanine, thymine, cytosine,and uracil, which are considered as the naturally occurring nucleotidesin relation to therapeutic and diagnostic application in humans.Nucleotides include the natural 2′-deoxy and 2′-hydroxyl sugars, e.g.,as described in Komberg and Baker, DNA Replication, 2nd Ed. (Freeman,San Francisco, 1992) as well as their analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotideshaving modified base moieties and/or modified sugar moieties (see e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443,Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M.,(1999) Biochemica et Biophysica Acta, 1489:117-139; Freier S. M., (1997)Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic AcidDrug Dev., 10:297-310); 2′-O, 3′-C-linked [3.2.0]bicycloarabinonucleosides. Such analogs include synthetic nucleotidesdesigned to enhance binding properties, e.g., duplex or triplexstability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15 M) of salts with inorganic cationssuch as Na+ or K+ (i.e., low ionic strength), temperature higher than20° C.-25° C. below the Tm of the oligomeric compound:target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). For example, the hybridization rate decreases 1.1% foreach 1% formamide. An example of a high stringency hybridizationcondition is 0.1. times. sodium chloride-sodium citrate buffer(SSC)/0.1% (w/v) SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleotides such that stable and specificbinding occurs between the oligomeric compound and a target nucleicacid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remainingnon-complementary nucleotides may be clustered or interspersed withcomplementary nucleotides and need not be contiguous to each other or tocomplementary nucleotides. As such, an antisense compound which is 18nucleotides in length having 4 (four) non-complementary nucleotideswhich are flanked by two regions of complete complementarity with thetarget nucleic acid would have 77.8% overall complementarity with thetarget nucleic acid and would thus fall within the scope of the presentinvention. Percent complementarity of an antisense compound with aregion of a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art. Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison, Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, Vol. 2, pp. 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

The terms “mammal”, “patient” or “subject” covers warm blooded mammalsthat are typically under medical care (e.g., humans and domesticatedanimals). Examples include feline, canine, equine, bovine, and human, aswell as only human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

In general, methods of administering compounds, including nucleic acids,are well known in the art. In particular, the routes of administrationalready in use for nucleic acid therapeutics, along with formulations incurrent use, provide preferred routes of administration and formulationfor the nucleic acids described above. Compositions disclosed herein canbe administered by a number of routes including, but not limited to:oral, intravenous, intracranial, intracerebro-ventricular,intraperitoneal, intramuscular, transdermal, subcutaneous, topical,sublingual, or rectal means. The disclosed compositions can also beadministered via liposomes or other nanoparticles (e.g., packagedmicrosomes). Such administration routes and appropriate formulations aregenerally known to those of skill in the art.

Accordingly, the subject invention provides methods of treatingneurological disease or disorder comprising administering an antagonistof Let-7i to a subject having a neurological disease or disorder. Invarious embodiments, the neurological disease or disorder is selectedfrom: severance of nerves or nerve damage, severance of cerebrospinalnerve cord (CNS) or CNS damage, damage to brain or nerve cells,traumatic brain injury, spinal cord injury, stroke, hypoxia, ischemia,brain injury, diabetic neuropathy, aging, neurodegenerative disease(such as Alzheimer's disease, Parkinson's disease or dementia),peripheral neuropathy, or peripheral nerve injury. Antagonists of Let-7iinclude antisense oligonucleotides, siRNA, shRNA, or interfering RNAthat down-regulate or inhibit Let-7i activity or function. Theinhibition of Let-7i function or activity can be mediated by degradationof the Let-7i miRNA when antisense oligonucleotides, siRNA, shRNA, orinterfering RNA specifically hybridize with Let-7i. In variousadditional embodiments, the disclosed methods of treatment can,optionally, include the method the administration of progesterone or acomposition thereof to said subject. The subject method can also furthercomprise the administration of BDNF to said subject. Antagonists ofLet-7i, progesterone, BDNF and compositions thereof can be administeredto a subject as independent compositions sequentially (e.g., acomposition comprising one of more Let-7i antagonist, a compositioncomprising progesterone and/or a composition comprising BDNF) or as acombined composition (i.e., a compositions comprising one or moreantagonist of Let-7i, progesterone, and/or BDNF).

Where the subject invention is used to treat signs of aging in asubject, for example, cognitive, behavioral and functional consequencesof aging in the nervous system are to be treated. These include, and arenot limited to: (a) changes in memory, (b) alterations of languagefunction, (c) visual-perceptual changes, (d) slowing of reaction time,and/or (e) decreased balance and coordination. One or more of theseconsequences of aging in the nervous system may be treated.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 General Methods

Generation of Primary Neuron- or Glia-enriched Cultures: The use ofanimals for the purpose of generating primary cultures was approved bythe Institutional Animal Care and Use Committee at the University ofNorth Texas Health Science Center. All mice will be handled according tothe Guide for the Care and Use of Laboratory Animals. Primary culturesof cortex and hippocampal neurons will be prepared from neonatal murinepups (C57BL/6 mice, Jackson Laboratory) as described by Sarkar et al.with modifications [22, 41]. Briefly, cortical tissues isolated fromnewborn mice (postnatal days 2-4, mixed gender) will be then dissociatedwith trypsin and DNase I for 10 min at 37° C., and wash twice withNeurobasal-A medium containing B-27 and further dissociated by gentletitration using a graded series of fine polished Pasteur pipettes. Aftercentrifugation at 200×g for 3 min at 4° C., dissociated cells will beresuspended in Neurobasal-A/B-27 medium, passed through a cell strainerwith 70 μm mesh, and plated at 1.0×105 cells/cm2 on culture dishesprecoated with poly-D-lysine. The culture dishes were kept at 37° C. inhumidified 95% air and 5% CO2. For primary neuron-enriched culture, theinitial culture medium was replaced after 5 h; subsequently, half of themedium was changed every 3 days. At day in vitro (DIV) 3,1-β-arabinofuranosylcytosine (AraC) was added to a final concentrationof 5 μM to prevent glial proliferation. Treatments of the primaryneuronal cultures started at DIV 14. For glial-enriched cultures,confluent mixed glial cultures were placed on the shaker for 48 hrs todislodge microglia, resulting in cultures enriched with astrocytepopulation.

Quantitative RT-PCR (microRNA): Total RNA was isolated from primaryastrocytes and mouse brains using the MiRNeasy Mini Kit (QIAGEN,Valencia, Calif.) according to the manufacturer's instructions.Concentrations of extracted RNA were determined using absorbance valuesat 260 nm. The purity of RNA was assessed by ratios of absorbance valuesat 260 and 280 nm (A260/A280 ratios of 1.9-2.0 were consideredacceptable). Total RNA (10 ng) was reverse transcribed into cDNA in atotal volume of 15 μl using the High-Capacity DNA Archive Kit (RocheApplied Science, Indianapolis, Ind.) according to the manufacturer'sinstructions. The reaction mixture contained water, 2× quantitative PCRMaster Mix (Eurogentec, Freemont, Calif.), and 20× Assay-On-Demand foreach target gene. A separate reaction mixture was prepared for theendogenous control, U6. The reaction mixture was aliquoted in a 96-wellplate, and cDNA added to give a final volume of 20 μl. Each sample wasanalyzed in triplicate. The comparative cycle threshold (Ct) method(2-ΔΔCt) was used to calculate the relative changes in target geneexpression.

BDNF Immuno Assay In situ: To define the amount of endogenous BDNFreleased, we will modify the ELISA in situ protocol developed byPromega. A 96-well Nunc MaxiSorp surface polystyrene flat-bottomimmunoplate was precoated with an anti-BDNF monoclonal antibody [diluted1:1,000 in coating buffer (25 mM sodium bicarbonate and 25 mM sodiumcarbonate, pH 9.7)]. After rinsing off unbound antibody with TBS-Tbuffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.05% (v/v) Tween-20]and blocking the plate to minimize nonspecific binding, the culturemedia was added to the plate for 2 hrs to equilibrate the cell growthenvironment. Primary astrocytes were then plated, and after a period oftime to ensure cell attachment to the plate, the appropriate treatmentswere applied. BDNF standards, ranging in concentration from 1.95 to 500pg/ml, was added in parallel wells. At the end of hormone treatment,cells were carefully washed with TBST. The plate was then incubated withthe polyclonal anti-human BDNF antibody. The amount of specificallybound polyclonal antibody was then detected through the use of theanti-IgY-horseradish peroxidase (HRP) tertiary antibody (finalconcentration=0.5 μg/mL), which when exposed to the chromogenicsubstrate (TMB reagent; Promega), changes color in proportion to theamount of BDNF present in the sample. The color intensity was quantifiedby measuring the absorbance at 450 nm with a Viktor3 ELISA plate reader(Perkin Elmer). Only values within the linear range of the standardcurve, and above the lowest standard, were considered valid. BDNF levelswere normalized to protein and are reported as a percentage of vehiclecontrol. This method allowed detection of as little as 2 pg/ml BDNFrelease in control cultures to ˜250 pg/ml in P4-treated cultures.

Ovariectomy: Mice will receive bilateral ovariectomy (OVX) using adorsal approach under isoflurane anesthesia. A small cut is made throughskin and abdominal muscles in left and right lateral abdomen. Thearteries to left and right ovaries will be ligated, and ovaries will becut. The muscles and skin will be sutured with 4-0 Vicryl absorbablesuture.

Implantation of flash-fused steroid pellets: Fused steroid pellets willbe made using the flash-melt method described by Ratka and Simpkins[40]. The pellets (containing P4 or control) will be implantedsubcutaneously into abdominal area.

Transient middle cerebral artery occlusion (MCAo): MCAo will be used toinduce transient focal cerebral ischemia (as previously described [41]).Briefly, under isoflurane anesthesia, a mid line incision will be madeon the neck. Common carotid artery (CCA), external carotid artery (ECA)and internal carotid artery (ICA) will be dissected from the connectivetissue. A silicon coated 6-0 nylon monofilament will be inserted intothe left ECA and advanced till it occludes the origin of MCA. The MCAwill be occluded for 60 minutes and then reperfusion attained bywithdrawing the suture.

ICV (intracerebroventricular) antagomir injections: ICV injections willbe performed as described by Sananbenesi et al. [42]. In brief, micewill be anaesthetized and affixed with a cannula ipsilateral to the sideof surgery (coordinates from Bregma: AP1/4_0.4 mm, L1/4_1.15 mm,V1/4_2.0 mm). Mice will receive a 0.5 uL infusion of Let-7i-silencingantagomir (Exiqon, Vedbaek, Denmark) or scrambled antagomir (5 ug), inartificial cerebrospinal fluid (Harvard Apparatus).

The pole test: Animals will be trained 2 days before MCAo procedure andwill be tested on day 3, 7, and 14 post stroke. Training will beachieved by placing animals facing downward on the pole and allow themto descend. After repeating this training 5 times, animals will then betrained in the regular turning and descending procedure. Mice will beplaced on the rod facing upward. Normally, animals will turn around andstart descending themselves. Those that do not, however, can beencouraged to turn by gently pushing to a side. After each trial, micewill be allowed to explore the cage for 15 s and then returned to theirhome cage. An interval of at least 5 min will be allowed between trials.Mice will tested 3 trials and average performance is recorded.

The wire hanging test: Animals will be trained 2 days before MCAoprocedure and will be tested on day 3, 7,and 14 post stroke. Animalswill be allow to suspend their bodies on a single wire stretched between2 posts 50 cm above the ground. Between the posts, a soft pillow will beplaced to avoid injury in case of a fall. Training will be achievedsimply with several rounds of habituation and trials. In the actualtesting phase, mice will be tested 3 times and average performance isrecorded as final values.

Statistical Analysis: We anticipate a minimal sample size (i.e., “n”) of4 per group in aim 1 studies and an “n” of 13 per group in aim 2studies. This number of sample size is based on the followingparameters: Detecting a minimal effect size of 20%, setting alpha=0.05,and a desired power of 80% or greater. Data (densitometric analysis forWestern blotting, or numerical data from cell viability assays and fromsynapse quantification assays) will be analyzed using analyses ofvariance (ANOVA) followed by analysis of differences between individualgroups using Tukey's post-hoc tests. Relative abundance of miRNA andmRNA transcripts will be evaluated using the 2^(−ΔΔCt) method [43].Resulting data will be analyzed using Dunnett's test to compare foldchange in the experimental groups relative to the control group.

Both purified BDNF and conditioned medium derived from P4-treatedastrocytes increased the expression of synaptic markers in neurons:Synaptogenesis has been considered as an important mechanism forfunctional recovery after stroke [21, 29, 30]. P4 has been shown toinduce synaptogenesis in various brain locations, including cortex andhippocampus [4, 31, 32]. Although the underlying mechanism remainsunclear, one potential mediator for P4-induced synaptogenesis is BDNF[33]. Our preliminary data showed that both conditioned media derivedfrom P4-treated mouse primary astrocyte cultures and purifiedrecombinant BDNF increased the expression of synaptophysin (apresynaptic terminal marker, usually overexpressed during the neuronalremodeling [34]) and GAP43 (a synaptic marker that is mainly synthesizedduring axonal outgrowth during neuronal development and regeneration[7]) in primary cortical neurons (FIGS. 1A-1B). Increased expressions ofboth markers have been linked to P4-induced synaptogenesis followingstroke [4]. In conjunction with our previous work demonstrating thatP4-induced BDNF release from glia is dependent on Pgrmc1[10], this datasupports that P4-induced increase in expression of synaptic markers ismediated, at least in part, by the Pgrmc1-dependent release of BDNF fromglia.

Overexpression of let-7i decreased Pgrmc1 and BDNF mRNA in primaryastrocytes: An in silico analysis, using three prediction softwareprograms (miRDB, TargetScan and microRNA.org), revealed putative Let-7binding sites in the 3′-UTR of Pgrmc1 and BDNF that were conserved inrat, mouse and human sequences. The Let-7 family of miRNAs includesmultiple evolutionarily conserved members (Let-7a, b, c, d, e, f, g, i;miR-98) that can exert similar functions [35]. Since it has beenreported that miRNA Let-7i directly binds to the 3′-untranslatedterminal region (UTR) of Pgrmc1 mRNA, thereby repressing Pgrmc1expression in a peripheral (non-CNS) cell type [36], we chose to focuson let-7i in this study and used another let-7 family member, let-7f, asa control for specificity. Our data show that in primary corticalastrocytes, an overexpression of the let-7i mimic (syntheticdouble-stranded miRNA-like RNA fragment), but not the let-7f mimic,resulted in decreased Pgrmc1 and BDNF mRNA levels (FIGS. 2A-2B),supporting the notion that Let-7i negatively regulates Pgrmcl/BDNFsystem in glia.

Overexpression of let-7i attenuated P4-induced BDNF release from primaryastrocytes: FIG. 3 demonstrates that overexpression of let-7i abolishedP4-induced BDNF release from primary cortical astrocytes. We previouslyshowed that P4 triggered significant release from glia by acting viaPgrmcl [10]. When considering data in both FIGS. 2A-2B and 3, theysupport our experimental model that states that let-7i inhibitsP4-induced BDNF release from glia by down-regulating expression of bothPgrmcl and BDNF.

Expression of Pgrmc1 and BDNF decrease as the function of age in mousebrain: Current literature lacks information regarding the effects of ageon the expression of Pgrmc1 within the brain. Interestingly, we found anage-associated decrease of Pgrmc1 and BDNF mRNA in mouse hippocampus(FIG. 4). The decline in Pgrmc1 level was noted in middle-aged mice, andpreceded the decrease of BDNF in old animals. Since Pgrmc1 is requiredfor P4-induced BDNF release from glia, decrease of Pgrmc1 expressionduring normal aging may dampen P4′s neuroprotective effect. Moreover,such a decline in Pgrmc1 (and BDNF) may also explain the increased riskfor stroke in older individuals.

A decreased expression of Pgrmcl correlates with an increased expressionof let-7i in the cerebral cortex following an experimentally-inducedischemic stroke: To determine the potential involvement of let-7i instroke, we induced an ischemic stroke in C57/B16 female mice using themethod of middle cerebral artery occlusion (MCAo), then we examinedexpression of the miRNA in the cerebral cortex 7 days post stroke. FIG.5 shows that, compared to sham group, expression of let-7i increases byabout 60% in stroked animals, which correlated with a decreased inPgrmc1 expression. Data from FIGS. 2A-2B and 5A-5B support ourconclusion that let-7i represses expression of Pgrmc1. The data alsosupport our use of the MCAo method as a suitable model to study theregulation of let-7i/Pgrmc1/BDNF axis in ischemic stroke.

Our data show that let-7i negatively regulates expression of Pgrmc1 andBDNF in primary astrocytes and there is an inverse correlation betweenlet-7i and BDNF/Pgrmc1 in the ischemic brain. Therefore, we anticipatedthat BDNF and Pgrmc1 will be elevated following intracerebro-ventricular(ICV) injection of anti-let-7i, relative to the scrambled control. P4 isknown to reduce infarct size, reverse functional deficits, and inducesynaptogenesis in experimental stroke models [3, 37]. Therefore, wepredicted that mice exposed to P4 will show an increase insynaptogenesis in the penumbra, smaller ischemic lesion and hence, apositive functional recovery (demonstrated by measures of motorfunction, to include the wire-hanging test). ICV injection of the Let-7iantagomir under conditions of stroke was thus, expected to reverse thesuppression of glial Pgrmc1/BDNF pathway, thereby, contributing to anenhanced P4-induced upregulation of synaptogenesis, smaller ischemiclesion and enhanced motor function. The scientific literature suggeststhat synaptogenesis in the penumbra significantly increases within hoursof stroke and can last for several weeks [23, 24, 38]. Therefore, weexamined a window of 0-14 day post MCAo to monitor synaptogenesis bothacute and intermediate time points. We used a published protocol for thedelivery of microRNA to the central nervous system [39], to ensure thatanti-let-7i ICV injection result in sufficient miRNA knock-down thatwould lead to an observable effect on synaptogenesis andneuroprotection.

FIG. 6 shows the effect of co-administration of the Let-7i antagomir andP4 on the stroke-induced lesion size. Areas of damaged/dead cells appearwhite, whereas live tissue appears red, as a function of metabolism ofthe TTC stain. Compared to animals that were not subject to sham surgery(i.e., all aspects of the surgery were conducted, except the occlusionof middle cerebral artery—1^(st) column of sections representing rostral(top most) to caudal (bottom most) aspects of the brain), the inductionof stroke (2^(nd) column of sections) showed obvious ischemic damage. P4had no statistically significant effect. Remarkably, the co-applicationof Let-7i and P4 led to a near complete protection from the ischemicstroke.

FIG. 7 shows the functional recovery in the same four groups of animalsdepicted in FIG. 6. Functional recovery of motor function, as defined byan assessment of grip strength, revealed that the combination of boththe Let-7i antagomir and P4 led to complete functional recovery 7 dayspost treatment.

EXAMPLE 2 Materials and Methods

Primary cultures: Dissociated cortical neurons were prepared andmaintained as previously described (44). Briefly, cortices were removedfrom neonatal mouse brains (postnatal day 2-4, mixed gender) anddissociated with 0.25% trypsin. Cortical neurons were then plated onglass coverslip or plastic culture dishes coated with poly-D-lysine(Sigma). The culture medium used was Neurobasal (ThermoFisherScientific), supplemented with Glutamax and B27 serum-free supplement(ThermoFisher Scientific). At day in vitro (DIV) 3, 5 μM finalconcentration of 1-β-arabinofuranosylcytosine (45) (Sigma) was added tothe neuronal cultures to prevent glial proliferation. Half of the mediumwas replaced with fresh medium every four days. For viability assay,cortical neurons were plated onto 96-well plates (Corning) at theconcentration of 1.2×10⁵ cells/cm². For immunocytochemistry, corticalneurons were plated onto 12 mm glass coverslip (Neuvitro) at the densityof 4×10⁴ cells/cm². Treatments of primary cortical neurons started atDIV12.

Primary cortical astrocytes were prepared and maintained as previouslydescribed (46), with some modifications. Briefly, cortices of post-natalday 2-4 mouse pups were dissociated with 0.25% trypsin and plated onto75 cm² tissue culture flask. The culture medium used was Dulbecco'smodified Eagle's medium (DMEM) (ThermoFisher Scientific), supplementedwith 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences) and10000U/ml Penicillin-Streptomycin (ThermoFisher Scientific). Afterreaching confluence, mixed glial cultures were placed on the shaker for48 h to dislodge microglia, resulting in cultures enriched withastrocyte population.

Treatment of primary cultures: To determine the miRNA regulation ofdownstream targets in primary cortical astrocytes, miRNA mimics andinhibitors were transfected into these cells for 48 hrs. Aftertransfection, total RNA and proteins were isolated for gene and proteinexpression analysis. Mock transfection was used as the control for theseexperiments.

To study the effect of miRNA on P4-induced BDNF release from astrocytes,BDNF in-situ ELISA were performed. Expression of miRNA was firstmanipulated by transfection as described above. 24 h after transfection,10 nM P4 was added to primary cortical astrocytes for additional 24 hwithout changing media containing transfection complexes. Vehiclecontrols were performed in parallel such that control cultures wereexposed to 0.1% dimethylsulfoxide (DMSO). The 10 nM concentration of P4used in studies described here was chosen because it has been reportedto elicit a maximal release of BDNF from astrocytes (10).

In experiments evaluating the effect of miRNA on P4-inducedneuroprotection and the synaptogenic marker, synaptophysin, we firsttransfected miRNA mimic and inhibitor into primary cortical astrocytesfor 24 h. Afterward, P4 (10 nM) was added to these cultures foradditional 24 h to generateP4-treated-astrocytes-derived-conditioned-media (P4-ACM). In parallel,treatment of 0.1% DMSO was performed to generateDMSO-treated-astrocytes-derived-conditioned-media (DMSO-ACM), whichserved as vehicle controls. Before applying to primary neurons, theseconditioned media were filtered through a 10 kD cut-off column toeliminate residual P4 and miRNA mimic or inhibitor. In neuroprotectionassay, astrocytes-conditioned-media were added to primary corticalneurons with prior exposure to one hour of oxygen-glucose-deprivation(OGD), an in-vitro model of ischemic-like insult. Based on ourexperience, 1 h of OGD was enough to induce 50% neuronal cell death.BDNF (50 ng/ml) was directly added to different groups after OGD toserve as positive control. Neuronal cultures exposed to normoxia wereused as the control for these data sets. 24 h after the applications ofBDNF or conditioned-media, CellTiter-Glo Luminescent cell viabilityassay (Promega) was performed to measure neuroprotection. Insynaptogenic marker measurement assay, BDNF andastrocytes-derived-conditioned-media were directly added to primarycortical neurons for 24 hrs. Synaptophysin expression and number ofsynaptophysin puncta in these neuronal cultures were assessed byimmunocytochemistry, followed by confocal imaging and analyzed usingImageJ (National Institutes of Health) software (47).

Transfection: Transfection of miRNA mimics and inhibitors was performedusing the Hiperfect transfection reagent (Qiagen) according tomanufacturer's instructions. Cells were transfected with miRNA mimicsand inhibitors for 48 h. This duration was chosen since it resulted inan optimal effect on targets-of-interest. Synthetic miRNA mimics(Syn-mmu-let-7i-5p, Syn-mmu-let-7f-5p) and inhibitors(Anti-mmu-let-7i-5p, Anti-mmu-let-7f-5p) were purchased from Qiagen.

Quantitative RT-PCR: Total RNA was isolated from primary corticalastrocytes and mouse brains using the MiRNeasy Mini Kit (Qiagen)according to the manufacturer's instructions. Concentrations ofextracted RNA were determined using absorbance values at 260 nm. Thepurity of RNA was assessed by ratios of absorbance values at 260 and 280nm (A260/A280 ratios of 1.9-2.0 were considered acceptable).

For miRNA expression measurements, total RNA (10 ng) was reversetranscribed into cDNA in a total volume of 15 μl using the microRNA cDNAArchive Kit (ThermoFisher Scientific) according to the manufacturer'sinstructions. The reaction mixture contained water, 2x quantitative PCRMaster Mix (Eurogentec), and 20× Assay-On-Demand for each target gene. Aseparate reaction mixture was prepared for the endogenous control, U6.The reaction mixture was aliquoted in a 96-well plate, and cDNA added togive a final volume of 20 μl. Each sample was analyzed in triplicate.The comparative cycle threshold (Ct) method (2^(ΔΔCt)) was used tocalculate the relative changes in target miRNA expression.

For mRNA expression measurements, total RNA (1.6 μg) was reversetranscribed into cDNA in a total volume of 20 μl using the High-CapacitycDNA Archive Kit (ThermoFisher Scientific) according to themanufacturer's instructions. The reaction mixture contained water, 2×quantitative PCR Master Mix (Eurogentec), and 20× Assay-On-Demand foreach target gene. A separate reaction mixture was prepared for theendogenous control, GAPDH. The reaction mixture was aliquoted in a96-well plate, and cDNA (30 ng RNA converted to cDNA) was added to givea final volume of 30 μl. Each sample was analyzed in triplicate.

The comparative cycle threshold (Ct) method (2^(−ΔΔCt)) was used tocalculate the relative changes in target gene expression.

PCR primers were purchased as Assay-On-Demand from ThermoFisherScientific. The assays were supplied as a 20 mix of PCR primers (900 nM)and TaqMan probes (200 nM). The let-7i (002221), U6 (001973), BDNF(Mm00432069_ml), GAP-43 (Mm00500404_ml), GAPDH (Mm99999915_gl), PSD-95(Mm00492193_ml), Pgrmc1 (Mm00443985_ml) and SYP (Mm00436850_ml) assayscontain FAM (6-carboxy-fluorescein phosphoramidite) dye label at the 5′end of the probes and minor groove binder and nonfluorescent quencher atthe 3′ end of the probes.

CellTiter-Glo Luminescent cell viability assay (Promega): This assayuses the level of adenosine triphosphate (48) as an indicator ofmetabolically active cells and is directly proportional to the number ofliving cells (49, 50). The assay was performed according tomanufacture's instruction. In brief, cell plate was first equilibratedto room temperature for 30 minutes. A volume of the kit reagent equal tothe volume of cell culture present was then added to each well. Theplate was then placed on an orbital shaker for 2 minutes to induce celllysis, followed by 10 minutes of incubation at room temperature.Luminescence was recorded using a plate reader.

BDNF Immuno Assay In situ: To determine the amount of endogenous BDNFreleased with P4 treatment, we performed ELISA in situ assay, aspreviously described (10). In brief, a 96-well Nunc MaxiSorp surfacepolystyrene flat-bottom immunoplate was precoated with an anti-BDNFmonoclonal antibody [diluted 1:1,000 in coating buffer (25 mM sodiumbicarbonate and 25 mM sodium carbonate, pH 9.7). After blockingnonspecific binding, primary cortical astrocytes were then plated,followed by appropriate treatments application. BDNF standards, rangingin concentration from 1.95 to 500 pg/ml, was added to parallel wells. Atthe end of hormone treatment, cells were carefully washed with TBST. Theplate was then incubated with the polyclonal anti-human BDNF antibody.The amount of specifically bound polyclonal antibody was then detectedthrough the use of the anti-IgY-horseradish peroxidase (HRP) tertiaryantibody, which when exposed to the chromogenic substrate (TMB reagent,Promega), changes color in proportion to the amount of BDNF present inthe sample. The color intensity was quantified by measuring theabsorbance at 450 nm with a Viktor3 ELISA plate reader (Perkin Elmer).Only values within the linear range of the standard curve, and above thelowest standard, were considered valid. This method allowed detection ofas little as 2 pg/ml BDNF release in control cultures to ˜250 pg/ml inP4-treated cultures.

Oxygen-glucose Deprivation (OGD): OGD was performed according to anestablished protocol, as described elsewhere, with minor modifications(51). Briefly, primary cortical neurons were carefully washed five timeswith Hank's balanced salt solution (HBSS, ThermoFisher Scientific) toremove residual glucose. Glucose-free DMEM (ThermoFisher Scientific) wasthen added to the cultures, and the plates were transferred into ahypoxic chamber (0.1% oxygen) for 1 h. At the end of hypoxia,glucose-free DMEM was replaced with regular maintaining media.Reoxygenation was initiated by transferring the cells to normoxic 5% CO2cell culture incubator.

Western blotting: Primary cortical astrocytes and mouse brains werelysed with RIPA lysis buffer containing protease and phosphataseinhibitors, as previously described (44). After homogenization, sampleswere centrifuged at 45,000 rpm for 30 min at 4° C. and supernatants werecollected. Total protein concentrations were determined using theBio-Rad DC protein assay kit (Bio-Rad Laboratories). Cell lysates wereseparated by SDS-PAGE and transferred onto polyvinylidene fluoridemembrane (Bio-Rad Laboratories) by electroblotting. Membranes wereblocked with 5% skim milk in tris-buffered saline containing 0.2% Tween20 (TBS-T) for lh at room temperature, followed by overnight incubationsof primary antibodies at 4° C. The following primary antibodies wereused: rabbit polyclonal anti-PSD 95 (1:1000, ab18258, Abcam), rabbitpolyclonal anti-Synaptophysin (1:1000, ab14692, Abcam), rabbitmonoclonal anti-GAP43 (1:200000, ab75810, Abcam), rabbit monoclonalanti-GAPDH (1:1000, 14C10, Cell Signaling), rabbit polyclonal anti-BDNF(1:300, sc546, Santa Cruz) and goat polyclonal anti-Pgrmc1 (1:500,ab48012, Abcam). After washing three times with TBS-T, membranes wereincubated with anti-goat IgG or anti-rabbit IgG conjugated withhorseradish peroxidase (Millipore) for 1 hr at room temperature. Aftertriple washes with TBS-T, immunoreactive bands were visualized with theECL detection system (ThermoFisher Scientific) and were captured using aluminescent image analyzer (Alpha Innotech). Densitometric analysis wasconducted using ImageJ (National Institutes of Health) software (47).

Immunofluorescence: The cortical neurons were fixed in 4%paraformaldehyde (45) for 15 min, followed by incubation in 0.2% TritonX-100 in Tris-buffered saline (TB S) for 15 min at room temperature forpermeabilization. Cultures were then blocked with 5% donkey serum/1%bovine serum albumin (BSA) in TBS for 1 h at room temperature andincubated with rabbit monoclonal anti-Synaptophysin (1:500, ab32127,Abcam) for 48 h at 4° C. After extensive rinsing with TBS-Tween 20,cultures were incubated with Alexa Fluor 647-conjugated secondaryantibody (1:500, Jackson ImmunoResearch Laboratories) for 2 h at roomtemperature. After extensive washing with TBS to remove unboundsecondary antibody, the coverslips were mounted onto glass slides (VWRScientific) using Vectashield mounting medium with DAPI (VectorLaboratories). The slides were observed under a confocal fluorescencemicroscope (FV1200, Olympus) with a 60× objective.

Mouse brains were fixed in 4% PFA overnight at 4° C. and subsequentlycryoprotected in 30% sucrose solution. The brains were then sectionedinto 40-μm thick coronal slices and subjected to immunostaining using anestablished protocol described elsewhere, with some modifications (52).In brief, brain sections were blocked in 5% donkey serum/1%BSA/TBSsolution for 2h at room temperature. In staining using mouse primaryantibody, sections were subsequently blocked in F(ab) fragment donkeyanti-mouse IgG (50 ug/ml, Jackson ImmunoResearch Laboratories) for 2 hat room temp to reduce background caused by secondary antibody bindingto endogenous mouse IgG in the tissue. After blocking step, brainsections were then incubated in primary antibody solution at 4° C. for72 h. Primary antibodies used were as follow: mouse monoclonal anti-NeuN(1:500, ab104224, Abcam); rabbit polyclonal anti-GFAP (1:1000, ab7260,Abcam); rabbit monoclonal anti-Synaptophysin (1:500, ab32127, Abcam) andgoat polyclonal anti-Pgrmc1 (1:200, ab48012, Abcam). Alexa Fluor 647,Alexa Fluor 594 or Rhodamine Red-conjugated secondary antibodies(Jackson ImmunoResearch Laboratories) were used at 1:500 dilution. Afterimmunostaining, sections were mounted onto microscope slides withVectashield mounting medium (Vector Laboratories) and observed under aconfocal fluorescence microscope (FV1200, Olympus) with a 63× objective.

Mice and treatments: All procedures with animals were reviewed andapproved by the Institutional Animal Care and Use Committee of theUniversity of North Texas Health Science Center. All institutional andfederal guidelines for the care and the use of animals were followed.Female C57BL/6J mice (18-week-old) were purchased from JacksonLaboratory. Animals were habituated to housing conditions one weekbefore experiments.

All mice were first ovariectomized to deplete endogenous ovarian hormonelevels. Two weeks after ovariectomy (OVX), P4 pellets weresubcutaneously implanted into these animals to replenish theirprogesterone levels. In parallel, different groups received cholesterolpellet implantations to serve as vehicle control. One week after pelletimplantation, stroke was induced in these mice using middle cerebralartery occlusion (MCAo) procedure. In parallel, different groupsreceived sham operation (non-stroke). 30 min after MCAo, 5 μg of eitherscrambled or let-7i inhibitor was injected into each animal brain viaintracerebroventricular (ICV) injection. Experimental groups includedsham-operated mice with cholesterol pellet implantation (sham), strokedmice with cholesterol pellet implantation and scrambled ICV injection(cholesterol+scrambled), stroked mice with P4 pellet implantation andscrambled ICV injection (P4+ scrambled), and stroked mice with P4 pelletimplantation and let-7i inhibitor ICV injection (P4 +anti-let-7i).

Ovariectomy: Bilateral ovariectomy (OVX) was performed using a dorsalapproach under isoflurane anesthesia, as described elsewhere (53).Briefly, small incisions were made bilaterally to expose ovaries. Thearteries adjacent to ovaries were ligated before ovaries removal.Incisions were then closed using 4-0 Vicryl absorbable suture.

Transient middle cerebralaArtery occlusion (MCAo): MCAo was performed toinduce transient focal cerebral ischemia, as previously described (54).In brief, mice were anesthetized with isoflurane inhalation. A mid-lineincision was made on the neck. Left common carotid artery (CCA),external carotid artery (55) and internal carotid artery (ICA) weredissected from the connective tissue. The left MCA was occluded by a 6-0monofilament suture (Doccol Corporation) introduced via internal carotidartery. After 45 minutes occlusion, the suture was withdrawn forreperfusion. In sham-operated animals, monofilament was advanced to MCAregion and withdraw immediately without MCA occlusion.

Intracerebroventricular (ICV) injection: 5 μg of either scrambled orlet-7i inhibitor (GE Healthcare Dharmacon) was suspended in 0.5 μL ofPBS and injected into lateral ventricles using a stereotaxic instrument,as previously described, with minor modifications (56). In brief, thesolution was injected using a 5-uL Hamilton syringe attached to theUltra Micro Pump UMP3 system (World Precision Instruments) at a flowrate of 0.2 μl/min. Coordinates used for ICV injection were AP −0.58 mm,ML+1.2 mm, DV −2.1 mm.

Assessment of brain tissue damage: 2,3,5-Triphenyltetrazolium chloride(TTC) staining: TTC staining was performed to assess ischemic injuryamong groups, as described in an established protocol (57). Briefly, 24h after MCAo, mouse brains were harvested and sectioned into 2-mm thickcoronal sections. These sections were immersed in 2% TTC solution for 30min at 37° C. and then fixed in 10% formalin. The stained slices werephotographed and subsequently measured for the surface area of theslices and the ischemic lesion (Image-Pro Plus 3.0.1, Silver Springs,Md., U.S.A.). Imaged of stained sections were captured and infarct sizeswere analyzed using ImageJ (National Institutes of Health) software(47).

Functional recovery assessment: wire suspension test: In ordered toassess motor function recovery with different treatments, wiresuspension test, a test of grip strength and endurance, was used, asdescribed elsewhere (58). In brief, mice were allowed to suspend theirbodies on a single wire that was elevated above a padded platform. Thelatency for animals to fall off the wire was recorded. Mice were trainedtwo days prior to MCAo to establish a baseline across groups. Trainingwas achieved with several rounds of habituation and trials. In theactual testing phase, each mouse was tested 3 times, and averageperformance was taken as final values. Performances of these mice wasevaluated at day 3, 7 and 14 post stroke.

Synaptophysin (SYP) optical density analysis and puncta quantification:For experiments using primary cortical neurons, mounted coverslips wereimaged using a confocal fluorescence microscope (FV1200, Olympus) with a63× objective. Healthy cells that were at least two cell diameters fromtheir nearest neighbor were identified and selected at random by eye byDAPI fluorescence. Ten non-overlapping fields per sample were imaged.Quantification of SYP immunoreactivity (IR) was performed using ImageJ(National Institutes of Health) software (47). Average IR was calculatedby dividing total IR value by the number of cells presented in thecaptured image. Synaptophysin puncta quantification was analyzed with acustom plug-in (written by Barry Wark; available upon request fromc.eroglu@cellbio.duke.edu) for ImageJ program. The details of thisimaging and quantification method can be found in a previous publication(59).

To quantify SYP fluorescence intensity and number of puncta in mousebrain, three independent coronal brain sections per animal were stainedwith SYP. 5-μm confocal scans were performed (optical section width,0.33 μm; 15 optical sections each) at 63× magnification, as previouslydescribed (60). Maximum projections of three consecutive opticalsections corresponding to 1-μm sections were analyzed by using theImageJ puncta analyzer option to quantify for numbers of SYP puncta (≥5optical sections per brain section and ≥15 total images per brain).Average SYP puncta density per imaged area was calculated for eachtreatment group.

Statistical Analysis: In vitro data obtained from no fewer than threeindependent experiments (where each independent experiment consisted ofbetween 5-8 replicates), and in vivo data obtained from at least 4animals per group (as many as 20 animals per group for the functionalrecovery/motor function tests) were analyzed using an analysis ofvariance (ANOVA), followed by appropriate post hoc analyses for theassessment of group differences, and presented as a bar graph depictingthe mean±S.E.M, using the GraphPad Software (San Diego, Calif.). Theparameters used to inform sample size considered the following:detecting an effect size of at least 30%, α=0.05, the variance of theendpoint measured, and achieving a statistical power of at least 0.8.

Let-7i antagomir inhibits oxygen-glucose-deprivation (OGD) inducedincrease in let-7i expression: Oxygen glucose deprivation (OGD), used inthe primary cortical astrocytes as an in vitro model of ischemia,revealed an increase in let-7i expression. Importantly, the data alsoverified the effectiveness of the let-7i antagomir to attenuate theOGD-induced increase in let-7i expression (FIG. 8A). The data in FIG. 8Bdemonstrate that OGD (which increases let-7i expression) compromised theability of progesterone (P4)-induced BDNF release from primary corticalastrocytes, similar to what was noted when let-7i was specificallyover-expressed.

Let-7i represses progesterone (P4)'s neuroprotection and its enhancementon synaptogenesis: To investigate the role of let-7i in P4'sneuroprotective effects, we manipulated miRNA expression in primarycortical astrocytes, then treated them with either vehicle (DMSO) or P4,following which astrocyte-derived conditioned media (ACM) was collected.The conditioned media was then applied to primary cortical neurons (daysin vitro (DIV)14) that had been exposed to oxygen-glucose deprivation(OGD). The neurons were then assessed for cell viability to ascertain ifconditioned media from P4-treated astrocytes elicited greaterneuroprotection relative to neurons treated with conditioned media fromDMSO-treated astrocytes (FIG. 9). We found that conditioned mediacollected from P4-treated astrocytes conferred similar neuroprotectionas seen in the positive control group (consisting of directadministration of BDNF (50 ng/ml) to the neuronal cultures). However,conditioned media collected from P4-treated astrocytes thatoverexpressed let-7i failed to promote the protection of neurons fromOGD.

Next, we determined if conditioned media from the different experimentalgroups represented in FIGS. 10A-10C resulted in changes in expression ofsynaptophysin, a presynaptic marker closely linked to synaptogenesis(4). We observed that conditioned media derived from P4-treatedastrocytes (P4-ACM) resulted in a robust increase in SYP (green)immunofluorescence (FIG. 10A), relative to neurons treated withconditioned media from DMSO-treated, and mock-transfected astrocytes.Quantitative analysis revealed that P4-ACM significantly increased bothSYP protein level (FIG. 10C) and the number of SYP puncta (FIG. 10B).The same observations were seen in the positive control group(consisting of direct application of BDNF (50 ng/ml) to the primaryneuronal cultures). Application of conditioned media collected fromP4-treated astrocytes that overexpressed let-7i (group label:let-7i+P4), however, failed to elicit the increase in synaptophysinexpression.

Combined treatment of progesterone (P4) and let-7i inhibition alleviateischemia-induced suppression of Pgrmc1 and BDNF expressions in thepenumbra of the ischemic brain: We next determined the expression oflet-7i in the middle cerebral artery occlusion model of ischemic stroke,focusing on changes in the penumbra. Assessments of let-7i expressionwere conducted at different time points—2, 7 and 14 days followingstroke. Representative images of immunoblots probed for Pgrmc1, alongwith pro-and mature-BDNF, are shown in FIG. 11A. We found that comparedto sham (non-stroked controls), ischemic injury resulted in anup-regulation of let-7i expression (FIG. 11E), starting at day 7 andremained elevated up to 14 days following stroke. P4 treatment alone(P4+a control sequence for let-7i (scrambled)) did not attenuate thestroke-induced increase in Let-7i. As expected,ischemia-induced-increase in let-7i expression was repressed in thegroup receiving combined treatment P4 and let-7i inhibition (P4+anti-7i)(FIG. 11E). Importantly, along with upregulating let-7i level, ischemiaalso resulted in a reduction of Pgrmcl protein level observed at day 7and day 14 (FIG. 11B). P4 treatment alone did not restore Pgrmc1 levelat either of the two time points. Combined treatment (P4+anti-let-7i),however, reversed ischemia-induced suppression of Pgrmcl protein levels.Furthermore, expression of mature BDNF was reduced as a consequence ofstroke at the 14 days post stroke evaluation period (FIG. 11D), whilepro-BDNF levels (FIG. 11C) remained unchanged across all time points andall treatments. Compared to sham, the treatment of P4 alone was able tomaintain the same level of mature BDNF, even at 14 days post stroke.Remarkably, combined treatment (P4+anti-let-7i) led to a robust increasein expression of mature BDNF observed at day 7 and day 14.

Combined treatment of progesterone (P4) and let-7i inhibition reducesischemic injury and enhances functional recovery: To examine the effectof P4 with or without the let-7i antagomir on the extent of ischemicinjury, we utilized 2,3,5-Triphenyltetrazolium chloride (TTC) stainingto visualize the size of the ischemic lesion. Representative images ofTTC stained are shown in FIG. 12A. Quantification of relative infarctsize (FIG. 12B) revealed that the combined treatment (P4+anti-let-7i)significantly reduced ischemic injury; whereas P4 treatment alone didnot.

Motor function (grip strength) was also evaluated using the wiresuspension test. Results (FIG. 13) showed that compared to the vehiclegroup (DMSO+scrambled), treatment of P4 led to a partial recovery ofmotor function, observed on day 7 and day 14. Interestingly, thecombined treatment of P4 and the let-7i antagomir resulted in a rapid,but partial, motor function recovery as early as 3 days post-treatment.By day 7, combined treatment led to complete functional recovery, andthe improvement was still evident at day 14. Results from FIGS. 12A-12Band FIG. 13 support our hypothesis that let-7i inhibition enhances P4'sneuroprotective effects that importantly, enhances functional recovery.

Inhibition of let-7i enhances progesterone (P4)'s effect on asynaptogenic marker: Synaptic plasticity in the ischemic penumbra regionhas long been known to influence the functional recovery after stroke(21, 23, 38). Therefore, to determine whether synaptogenesis occurringin the penumbra could be a factor contributing to functional recoveryobserved in FIG. 13, we extended our in vitro findings, to evaluate theexpression of synaptophysin (SYP), a synaptogenic marker, in thepenumbra of stroked mice. To do so, we performed immunofluorescence tovisualize SYP expression (red) (FIG. 14A) and quantified the relativenumber of SYP puncta, which is an indication of potential synapses (FIG.14B). In parallel, Western blot analysis was performed to evaluate totalSYP protein levels. Representative immunoblots probed for SYP are shownin FIG. 14C, and its relative quantification of protein level isdepicted in FIG. 14D. Results revealed that ischemia resulted in asustained downregulation of synaptophysin puncta (FIG. 14B) in thepenumbra at day 2,7 and 14 post-stroke. In addition, ischemic injury ledto decreased SYP protein level at day 2 and 14. There was a transientincrease in SYP expression at day 7, which could be due to acompensatory response to the ischemic injury. P4 treatment alone led toa delayed, but sustained, restoration in SYP total protein expression,observed at day 7 and day 14. With regards to the number of SYP puncta,the positive effect of P4 was only evident at day 14 post-treatment.Interestingly, at day 7 and 14, combined treatment (P4+anti-let-7i)resulted in significantly higher expression of SYP, compared to shamcontrols and P4 treatment alone. This combined treatment also led to acomplete restoration of synaptophysin puncta at day 7, an effect thatwas further enhanced at day 14. Taken together, these findings indicatethat P4 induces synaptogenesis in the penumbra of ischemic brain andthat let-7i inhibition further enhances this beneficial function of P4.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

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We claim:
 1. A method of treating a neurological disease or disordercomprising administering one or more antagonist of Let-7i or acomposition comprising said Let-7i antagonist to a subject having aneurological disease or disorder.
 2. The method according to claim 1,wherein the neurological disease or disorder is selected from: severanceof nerves or nerve damage, severance of cerebrospinal nerve cord (CNS),damage to brain or nerve cells, traumatic brain injury, spinal cordinjury, hypoxia, ischemia, brain injury, diabetic neuropathy, peripheralneuropathy, aging, neurodegenerative disease, or peripheral nerveinjury.
 3. The method according to claim 2, wherein said neurologicaldisease or disorder is aging of the nervous system or an age-associatedneurodegenerative disease.
 4. The method according to claim 3, whereinsaid aging of the nervous system comprises one or more of the following:(a) changes in memory, (b) alterations of language function, (c)visual-perceptual changes, (d) slowing of reaction time, and/or (f)decreased balance and coordination.
 5. The method according to claim 1,wherein said antagonist of Let-7i is an antisense oligonucleotide,siRNA, shRNA, or interfering RNA that down-regulates Let-7i activity orfunction.
 6. The method according to claim 1, said method furthercomprising the administration of progesterone or a composition thereofto said subject.
 7. The method according to claim 1, said method furthercomprising the administration of BDNF or a composition thereof to saidsubject.
 8. The method according to claim 6, wherein said antagonist ofLet-7i or composition thereof and/or said progesterone or a compositionthereof is/are administered separately, concurrently, or as a singlecomposition.
 9. The method according to claim 7, wherein said antagonistof Let-7i or a composition thereof and/or said BDNF or compositionthereof is/are administered separately, concurrently, or as a singlecomposition.